Integrins, extracellular matrix molecules, cytoskeletal proteins, and regulators such as Btbd7 contribute to cell migration and signaling by complex, integrated mechanisms. We are addressing the following specific questions: 1. What subcellular structures and signaling pathways are important for efficient cell migration in two-dimensional and three-dimensional environments? 2. How are the functions of integrins, the extracellular matrix, and the cytoskeleton integrated, and how is the regulatory crosstalk between them coordinated to produce effective cell migration? We are using a variety of cell and molecular biology approaches to address these questions, including biochemical analyses, fluorescent chimeras, live-cell phase-contrast, confocal, and two-photon time-lapse microscopy, as well as methods for evaluating the local matrix responses to an individual migrating mammalian cell. We have generated a variety of fluorescent molecular chimeras and mutants of cytoskeletal proteins as part of a long-term program to analyze their functions in integrin-mediated processes. We have been focusing particularly on the functions and regulation of integrins and associated extracellular and intracellular molecules in the mechanisms and spatial governance of cell migration. The developmentally crucial migration of neural crest cells to form craniofacial structures during embryonic development depends on the extracellular matrix protein anosmin. Genetic defects in anosmin result in human Kallmann syndrome. We and others previously established that anosmin functions in neural crest formation, cell adhesion, and neuronal migration. We recently established cell adhesion and spreading assays for anosmin and used them for antibody inhibition analyses to search for hypothesized integrin adhesion receptors. We find that the integrins alpha5-beta1, alpha4-beta1, and alpha9-beta1 are required for effective human cell adhesive receptor function in cell adhesion and cell spreading on anosmin. In addition, cell adhesion to anosmin can be inhibited by certain peptides: RGD peptides known to affect interactions with alpha5-beta1, and CS1-based peptides known to disrupt adhesion to alpha4-beta1. This identification of the integrin adhesion receptors used by anosmin should facilitate further studies of this matrix protein altered in Kallmann syndrome. Cell migration is also a prominent feature of cellular behavior during the establishment of tissue organization in embryonic development, mammalian branching morphogenesis, and adult wound healing. For non-biased, in-depth characterization of cell migration involving large numbers of cells, a computer-based system was developed that uses algorithms for video fluorescence microscopy tracking of Hoechst-labeled nuclei of cells to provide rapid quantification of speed, persistence, and other features of directional or random migration. This program termed FastTracks was initially developed using MATLAB software, and it can now be downloaded for use on regular computers without proprietary software. Using cell migration analyses based on computer-assisted individual cell tracking in vitro and in vivo, we recently found that the newly characterized regulator Btbd7 is essential for the two-fold increased rate of migration of peripheral epithelial cells in developing mouse salivary glands. A significant portion of these effects of Btbd7 on cell motility are due to its non-transcriptional down-regulation of E-cadherin protein levels. These enhanced dynamic cell movements appear to be crucial for normal in vivo cleft formation and branching morphogenesis of several mammalian organs. We are currently attempting to establish an immortalized human cell model for dissecting the in vitro functions of Btbd7 in cell-matrix interactions. This combined approach involving characterization of the regulation of cell migration and phenotypes in various microenvironments should provide novel approaches to understanding, preventing, or ameliorating migratory processes used by cells during abnormal embryonic development and particularly in cancer. An in-depth understanding of the precise manner in which cells move and interact with their matrix environment will also facilitate tissue engineering studies.